The present invention relates to a method to enhance the efficacy of a vaccine by administration of a chemokine in conjunction with the vaccine. The present invention also relates to compositions useful in the practice of this method.
DNA immunization is presently being developed as an inexpensive and safe means for providing immunizations to large numbers of people. One of the shortcomings of this approach is the relatively weak immune response triggered by DNA vaccines. The present invention provides for the administration of select chemokines to enhance the immune response to DNA vaccines.
Because of their ability to attract discrete sub-populations of leukocytes to sites of inflammation and antigen presentation, chemokines are key agents in eliciting immune responses. In addition, chemokines reportedly can influence the T helper response towards a Th1 (i.e. cell mediated) or Th2 (i.e. humoral) profile, according to the differential distribution of receptors on target cells.
Preferred chemokines according to the practice of the present invention include RANTES (regulated on activation, normal T cell expressed and secreted), a chemokine which has receptors on both Th1 and Th2 cells, MCP-1 (monocyte chemoattractant protein), which is Th1-type associated, and the Th2-type associated chemokine BLC (B lymphocyte chemoattractant) and MDC (macrophage-derived chemoattractant). The inventors have studied the immuno-modulating properties of these chemokines in conjunction with DNA immunization, using HIV-1BaL gp 120 and membrane-bound HIV-1BaL gp 160 as antigens.
The inventors have discovered that chemokines modulate immune responses according to their Th-type polarization. Accordingly, the investigated chemokines can be ranked in the following order of induction of Th-2 vs Th-1 responses: BLC (inducing mostly humoral responses), MDC, MCP-1 and RANTES (inducing mostly cellular responses). Quantitatively, MCP-1 was the strongest inducer of cellular responses, while the BLC induced the strongest humoral response.
These results are of great importance from the perspective of developing and optimizing vaccination regimens, in particular, against viral infections or cancer, but also, in general where directed or combined Th-type responses are sought.
DNA-based vaccines combine safety, ease of use, handling and modification and cost-effective production with the efficacy and effectiveness of live-attenuated vaccines. They are capable of eliciting both strong humoral and cell-mediated immunity (Moelling, K., 1997, Donnelly, J. J. et al., 1997; Montgomery, D. L. et al., 1997). Therefore DNA immunization represents a new approach for prevention (vaccination) and treatment (immune-based therapy) of infectious and neoplastic diseases. It has been shown that many factors contribute to the outcome of the immune response (Cohen, A. D. et al., 1998; Chun, S. et al., 1998) and might therefore be exploited. The present inventors have discovered that co-administration of chemokines with DNA vaccines significantly affects the immune response, both in amplitude and quality. The chemokines used in the empirical work presented herein are representative of classes of chemokines which attract specific subsets of lymphocytes and/or antigen presenting cells.
The surprising discovery that genes in plasmid expression vectors are expressed in vivo after intramuscular (i.m.) injection and that this expression stimulates an immune response against the plasmid-encoded proteins, has led to the concept of xe2x80x98DNA vaccinationxe2x80x99 (Wolff et al., 1990). It has been shown that upon plasmid injection both antigen-specific antibody (Ab) responses and cytotoxic T Cell (CTL) responses are produced (Tang, D., 1992; Wang et al., 1993; Ulmer et al., 1993), without damaging the muscular tissue. The DNA vaccination procedure is safe because it uses only a part of the genome of the pathogen, hence making an active infection impossible. The ability to express virtually any antigen, or antigen combination by genetic engineering, coupled with efficacy and safety, has fueled the great popularity of DNA vaccines.
A considerable number of studies have focused on HIV and cancer vaccines (Fomsgaard, A., 1999; Barnett, S. W. et al., 1998; Kennedy, R. C., 1997; Oppenheim, J. et al., 1996; Burton, D. R. and Moore, J. P., 1998), and phase III studies using HIV-based vaccines have already begun (VaxGen (1999). However, current DNA vaccines still need improvement, since it is likely that both strong CTL responses and high neutralizing Ab titers are required, at least for some vaccines to be effective (Corel, et al., 1998), and indeed, most of the vaccines used have only limited efficacy in inducing efficient humoral responses (AIDS Alert, 1998).
DNA immunization is generally performed using immuno-stimulants (adjuvants) (Falo, L. D. Jr. and Storkus, W. J., 1998, Sasaki, S., 1998). Those adjuvants can be of very different nature (Allison, A., 1997; Gupter, R. K. and Siber, G. R., 1995; Cox, J. C. and Coulter, A. R., 1997): currently used preparations include bacterial cell-wall derivatives (Freund""s adjuvant), oil-based emulsions (MF-51, SAF-1), aluminum salts (alum), saponine derivatives (QS21), or polymers (polyphosphazene). More recently cytokines and chemokines have been proposed as xe2x80x9cnaturalxe2x80x9d adjuvants, in both DNA-based and traditional immunization protocols (Xin, K. Q., 1999; Sin, J. I. et al 1999; Wang, B. et al., 1993; Okada, E. et al., 1997).
Classical protein vaccination protocols are effective in inducing high humoral response (Sha, Z. et al., 1999), unlike, for the time being, DNA immunization. The immunization with protein also allows the establishment of an accurate dose dependency, which is not possible using DNA vaccination protocols, since in DNA vaccination the ratio of administrated and finally expressed DNA remains unknown. On the contrary, the exact amount of injected protein is known and there is no delay via the process of transcription, translation and secretion, so that when chemokines are coadministered, their potential enhancing effect on antigen presentation will occur in hours rather than days.
Protein immunization is not very effective for inducing cell-mediated immunity, due to the route of antigen processing and presentation. However, depending on the pathogen, cellular responses may be crucial (Connor, R. I. et al., 1998). Special adjuvants have been designed to circumvent this hurdle (Sheikh, N. A. et al., 1999); however, these formulations using these adjuvants have the potential disadvantage of denaturing the protein, and thereby possibly preventing the elicitation of a relevant antibody response (VanCott, T. C. et al., 1997).
Another advantage of protein immunization lies in the ability to quickly study potential B or T cell epitopes. Peptide vaccination has been shown to be promising in both T cell induction and suppression (Ruiz, P. J., 1999: Toes, R. E., 1996), which has lead to extensive studies in pharmaceutical drug design, although T cell epitopes, in contrast to B cell epitopes, would be limited to the major MHC-haplotypes that present them.
Protein vaccination is as safe as DNA vaccination since there is no risk of infection by a live pathogen. In addition, autoimmune or tolerance reactions, which might be elicited by long-term expression of plasmid-based vaccines (Mor, G., 1997), are less likely to be induced by protein immunization. Therefore, protein-DNA mixed protocols have the potential of combining the advantages of DNA and protein immunization, with the lowest risk of inducing undesired or ineffective responses (Bruehl, P. et al., 1998; Richmond, J. F. L. et al., 1998; Barnett, S. W., 1998).
Chemokines are a family of small cytokines, that are released in response to infection together with other inflammatory cytokines (Mackay, C. R., 1997). Their molecular masses range from 6-14 kDa (Ward, S. G., 1998), and they all have related amino acid sequences which are between 20 and 50% sequentially homologous. Chemokines are multiple mediators, but were first studied as inducers of chemotaxis of specific leukocytes (Nelson, P. I. and Krensky, E. M., 1998; Kim, C. H. et al., 1999; Moser B, 1998). Further studies have revealed that chemokines also stimulate lymphocyte development, angiogenesis, degranulation of granulocytes, respiratory bursts and the release of lysosomal enzymes in monocytes. Furthermore, chemokines were shown to reduce the threshold of responsiveness of immune cells to other inflammatory mediators (Taub, D. D., 1996). These properties render chemokines particularly important in localizing and enhancing inflammation.
Chemokines are divided into four different subfamilies, according to the position of the first two cysteines in their primary sequence: the xcex1-chemokine subclass bears a CXC-motif, where the two cysteines (C) are separated by one amino acid; the xcex2-chemokines contain a CC motif, the xcex3-subclass lacks one cysteine residue, and is yet represented only by one member, lymphotactin, and in xcex4-chemokines, or CX3C subclass, the two cysteines are separated by three amino-acids. These cysteine residues form disulfide bridges with two other cysteines located further downstream in the primary sequence, thus stabilizing the tertiary structure of these chemokines.
Both primary and tertiary structures are crucial in inducing differential chemoattractant responses, determining binding to specific receptors (Baggiolini, M., 1997). However, the chemokine/chemokine receptor system is in part redundant, since overlapping ligand specificity by interaction with several chemokine receptors has been shown (Campbell, J. J. et al., 1997). This relatively complicated network of ligand/receptor interaction has been tentatively explained by the xe2x80x9cmultistep navigation modelxe2x80x9d (Foxman, F. F., 1997), according to which migrating lymphocytes follow a hierarchy, of chemotactic gradients. Leukocytes will encounter and respond to multiple chemoattractant signals in a complex spatial and temporal pattern. Those signals guide the leukocytes from the vascular system to the site of inflammation by inducing sequentially their rolling through the endothelium and the firm adhesion and extravasation at the peak of the gradient, namely, where most adhesion molecules are expressed. Additional leukocytes may be directed via other signals through the tissue to the site of inflammation. During this migration, several receptors may be triggered simultaneously and/or successively on both leukocytes as on the cells involved in their xe2x80x98guidance.xe2x80x99 This model therefore proposes specific blockage sites for therapeutic purposes, such as treating allergic and autoimmune diseases, with fewer side effects than common immunosuppressants. Specific blocking of chemokine-receptors could be achieved by specific antagonists, such as monoclonal Abs or modified chemokine derivates (Simmons, G. et al., 1997; Chen, J. D., 1997; Baggliolini, M. and Moser, B., 1997; Wu, L., 1997).
Chemokine receptors are designated CXCR followed by a number when binding cc-chemokines and CCR followed by a number when binding xcex2-chemokines. All chemokine receptors belong to the group of G-protein-coupled seven transmembrane domain receptors (Baggiolini M, 1997).
Human immunodeficiency virus (HIV) induces a persistent and progressive infection leading, in the vast majority of cases, to the development of the acquired immunodeficiency syndrome (AIDS) (Barre-Sinoussi et al., 1983, Science 220:868-870; Gallo et al., 1984, Science 224:500-503). The HIV envelope surface glycoproteins are synthesized as a single 160 kilodalton precursor protein, which is cleaved by a cellular protease during viral budding into two glycoproteins, gp41 and gp120. gp41 is a transmembrane glycoprotein and gp120 is an extracellular glycoprotein which remains non-covalently associated with gp41, possibly in a trimeric or multimeric form (Hammerskjold, M. and Rekosh, D., 1989, Biochem. Biophys. Acta 989:269-280). The V3 loop of gp120is the major determinant of sensitivity to chemokine inhibition of infection or replication (Cocchi et al., 1996, Nature Medicine 2:1244-1247; and Oravecz et al., 1996, J. Immunol. 157:1329-1332).
Although considerable effort is being directed towards the design of effective therapeutics, currently no completely curative anti-retroviral drugs against AIDS exist. The HIV-1 envelope proteins (gp160, gp120, gp41) have been shown to be the major antigens for neutralizing anti-HIV antibodies present in AIDS patients (Barin et al., 1985, Science 228:1094-1096). These proteins are promising antigen candidates for anti-HIV vaccines. Several groups have begun to use various portions of gp160, gp 120, and/or gp41 as immunogenic targets for the host immune system (see, for example, Ivanoff, et al., U.S. Pat. No. 5,141,867; Saith, et al., PCT publication WO 92/22654; Shafferman, A., PCT Publication WO 91/09872; Formoso, et al., PCT Publication WO 90/07119). Therefore, methods to increase the efficacy of vaccines against HIV, especially vaccines using gp120 as the antigen, are needed.
Additionally, a novel vaccine technology, designated genetic vaccination, nucleic acid vaccination or DNA vaccination, has been explored to induce immune responses in vivo. Injection of cDNA expression cassettes results in in vivo expression of the encoded proteins (Dubensky, et al., 1984, Proc. Natl. Acad. Sci. USA 81:7529-7533; Raz, et al., 1993, Proc. Natl. Acad. Sci. USA 90:4523; Wolff, et al., 1990, Science 247:1465-1468), with the concomitant development of specific cellular and humoral immune responses directed against the encoded antigen(s) (Wang, et al., 1995, Hum. Gene Ther. 6:407-418; Ulmer, et al., 1993, Science 259:1745-1749; Tang, et al., 1992, Nature 356:152-154; Michel, et al., 1995, Proc. Natl. Acad. Sci. USA 92:5307-5311; and Lowrie, et al., 1994, Vaccine 12:1537-1540). Humoral and cellular responses have been induced to HIV-1 and SIV antigens through various applications of this technology in macaques (Wang, et al., 1995, Virology 221:102-112; Wang, et al., 1993, Proc. Natl. Acad. Sci. USA 90:4156-4160; and Boyer, et al., 1996, J. Med. Primatol. 25:242-250) as well as mice (Wang, et al., 1995, Virology 221:102-112; Lu, et al., 1995, Virology 209:147-154; Haynes, et al., 1994, AIDS Res. Hum. Retroviruses 10 (Suppl. 2):S43-S45; Okuda, et al., 1995, AIDS Res. Hum. Retroviruses 11:933-943).
Recently, Lekutis, et al. (1997, J. Immunol. 158:4471-4477), assessed the TH cell response elicited by an HIV-1 gp120 DNA vaccine in rhesus monkeys by isolation of gp120-specific, MHC class II-restricted CD4+ T cell lines from the vaccinated animals. Lekutis, et al. showed that the isolated cell lines proliferated in response to APC in the presence of recombinant gp120, as well as to APC expressing HIV encoded env protein. Lekutis, et al. further showed that these cell lines responded to env by secreting IFN-xcex93 and IFN-xcex1without appreciable IL-4 production. These results demonstrate that the animals exhibited a cellular immune response to the DNA vaccine.
Boyer, et al. (1997, Nature Medicine 3:625-532), inoculated chimpanzees with an HIV-1 DNA vaccine encoding Env, Rev, and Gag/Pol, and found that the immunized animals developed specific cellular and humoral immune responses to these proteins. After challenging the immunized animals with a heterologous chimpanzee titered stock of HIV-1 SF2, Boyer, et al. further found, using a Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assay, that those animals vaccinated with the DNA vaccine were protected against infection whereas the control animals were not so protected.
Kim, et al., (1997 J. Immunol. 158:816-826), investigated the role of co-delivery of genes for IL-12 and GM-CSF along with DNA vaccine formulation for HIV-1 antigens env and gag/pol in mice. Kim, et al. observed a dramatic increase in specific CTL response from the mice immunized with the HIV-1 DNA vaccine and IL-12. Kim et al. also observed that the co-delivery of IL-12 genes resulted in the reduction of specific antibody response, whereas the codelivery of GM-CSF genes resulted in the enhancement of specific antibody response. Kim, et al. further observed that co-delivery of IL-12 gene with a HIV DNA vaccine results in splenomegaly (Kim, et al. 1997, J. Immunol., 158:816-826), which has been shown in mice to have toxic effects such as weight reduction or even death (Eng, et al., 1995, J. Exp. Med. 181:1893; Stevensen, et al., 1995, J. Immunol. 155:2545; and Orange, et al., 1995, J. Exp. Med. 181:901).
Notwithstanding the recent developments in the field of HIV DNA vaccines, there still exists a need for a method to enhance the efficacy of a vaccine, especially an HIV DNA vaccine. For instance, both cellular and humoral immune responses are needed to control an HIV-1 infection (Boyer, et al., 1997, Nature Medicine 3:625-532). The induction of both cellular and humoral immune response by the Berjer, et al. method is still quite low because only one of the three immunized chimpanzees developed both cellular and humoral responses. Similarly, although co-delivery of an IL-12 encoding gene with a HIV DNA vaccine, as described in Kim, et al. (1997, J. Immun. 158:816-826), may have enhanced the cellular immune response, this co-delivery also decreased the humoral response.
Citation of any of the references discussed hereinabove shall not be construed as an admission that such reference is prior art to the present invention.
The present invention relates generally to the use of chemokines, particularly chemokines which attract lymphocytes or antigen presenting cells, for enhancing an immune response to an antigen, particularly to a vaccine antigen.
In one aspect, the invention relates to a method for enhancing the efficacy of a vaccine in a subject. The method generally comprises administering to the subject: a first component selected from the group consisting of: (i) an antigen against which an immune response is desired in the subject; and (ii) a nucleic acid encoding the antigen of (i). In addition to the first component, the subject is also administered a second component selected from the group consisting of: (i) a chemokine selected from the group consisting of RANTES, MCP-1, MDC and BLC, or a functional equivalent (as defined herein) of said chemokine; and (ii) a nucleic acid encoding the chemokine of (i). The first component and second component are administered in an immunizingly effective amount (as defined herein).
In one aspect of the invention, the second component comprises a human chemokine or a functional equivalent thereof.
In another aspect of the invention, the second component is administered concurrently with the first component. The second component may also be administered within a time period before or after administration of the first component, which time period is sufficient to achieve enhancement of the efficacy of the vaccine.
The first component may be any of a wide variety of antigens known in the art. However, in a preferred embodiment of the invention, the first component is an HIV antigen. A preferred HIV antigen is a gp120 antigen, which in addition to a native gp120, also includes analogs, derivatives and fragments of gp120 which produce an antibody response (i.e., polypeptides which are functionally equivalent in that they produce an antibody response wherein the antibodies produced by such response will bind to native gp120), which antibodies also have specificity for a native HIV gp120.
In another aspect, the invention relates to a nucleic acid encoding both the first component and the second component.
In a method aspect of the invention, the first component and the second component are provided as nucleic acid sequences on the same or on separate nucleic acids and are administered directly to the subject. The first component and the second component may also be provided as nucleic acid sequences on the same or on separate nucleic acids and may be used to transform a cell, which cell is administered to the subject. The first component suitably comprises a nucleic acid encoding an HIV antigen, preferably a gp120 antigen. The subject is preferably a human and may be HIV positive or may exhibit behavioral patterns or occupational factors associated with risk of becoming HIV positive.
In another aspect, the invention relates to a method for improving the speed of an antibody response to a soluble antigen in a subject, comprising co-administering to the subject the soluble antigen with BLC. The soluble antigen is preferably an HIV antigen, more preferably a gp120 antigen. The subject is preferably a human.
The invention also provides a method for reducing the number of immunizations in an immunization regimen required to achieve an improvement in a subject""s immune response to an antigen. This method generally comprises administering MDC and the antigen to the subject. The antigen may suitably be administered before, during or after the administration of MDC. However, the antigen is administered in sufficient temporal proximity to the administration of MDC to achieve an improvement in the subject""s immune response to the antigen. The antigen is preferably an HIV antigen, and the subject is preferably a human.
In another aspect, the invention provides a method for suppressing an immune response in a subject in need thereof. This method generally comprises administering to the subject an amount of an MCP-1 antagonist which, in comparison to a corresponding immune response in the absence of the MCP-1, suppresses the immune response.
In yet another aspect, the invention provides a method for inducing or enhancing a humoral response in a subject in need thereof, the method comprising administering to the subject a humoral response-inducing amount of BLC.
The invention also relates to a method for inducing a subject to produce MIP-1xcex1. This method aspect of the invention generally comprises administering to the subject an MIP-1xcex1-inducing amount of RANTES. The subject is preferably HIV positive.
The invention also relates to compositions for achieving the various method aspects of the invention. For example, in one aspect, the invention relates to a composition comprising a first component selected from the group consisting of: (i) an antigen against which an immune response is desired in the subject, and (ii) a nucleic acid encoding the antigen of (i); along with a second component selected from the group consisting of: (i) a chemokine selected from the group consisting of RANTES, MCP-1, MDC and BLC, or a functional equivalent of said chemokine, and (ii) a nucleic acid encoding the chemokine of (i). This composition preferably also comprises one or more of each of the following pharmaceutically acceptable components: carriers; excipients; auxiliary substances; adjuvants; wetting agents; emulsifying agents; pH buffering agents; and other components known for use in vaccine or other pharmaceutical compositions.
The invention also relates to a nucleic acid comprising: a first nucleic acid sequence encoding an antigen against which an immune response is desired in the subject; and a second nucleic acid sequence encoding a chemokine selected from the group consisting of RANTES, MCP-1, MDC and BLC, or a functional equivalent of said chemokine. The first and second nucleic acid sequences are preferably expressed in a coordinated manner upon introduction into a subject to produce an amount of the first component that is immunogenic and an amount of the second component that is effective to enhance the efficacy of the vaccine. A related aspect of the invention involves the administration of this nucleic acid to a subject in need thereof to elicit an immune response to the antigen. The nucleic acid is suitably administered as a component of a pharmaceutical composition and may be administered directly to the subject and/or introduced into a suitable host cell and said suitable host cell is administered to the subject. The host cell may be obtained from the subject or from a cell culture originating from one or more cells obtained from the subject.
In a preferred embodiment, the invention provides a method for enhance the efficacy of an HIV vaccine. A preferred polypeptide for use with the HIV vaccine is gp120.
As used herein, a xe2x80x9cpharmaceutically acceptablexe2x80x9d component (such as a salt, carrier, excipient or diluent) of a formulation according to the present invention is a component which (1) is compatible with the other ingredients of the formulation in that it can be combined with the active ingredients (e.g. chemokine and/or antigen) of the invention without eliminating the biological activity of the active ingredients; and (2) is suitable for use in animals (including humans) without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are xe2x80x9cunduexe2x80x9d when their risk outweighs the benefit provided by the pharmaceutical composition.
The term xe2x80x9cimmunizingly effectivexe2x80x9d is used herein to refer to an immune response which confers immunological cellular memory upon the subject, with the effect that a secondary response (to the same or a similar antigen) is characterized by one or more of the following characteristics: shorter lag phase in comparison to the lag phase resulting from a corresponding exposure in the absence of immunization; production of antibody which continues for a longer period than production of antibody for a corresponding exposure in the absence of such immunization; a change in the type and quality of antibody produced in comparison to the type and quality of antibody produced from such an exposure in the absence of immunization; a shift in class response, with IgG antibodies appearing in higher concentrations and with greater persistence than IgM; an increased average affinity (binding constant) of the antibodies for the antigen in comparison with the average affinity of antibodies for the antigen from such an exposure in the absence of immunization; and/or other characteristics known in the art to characterize a secondary immune response.
The term xe2x80x9cfunctional equivalentxe2x80x9d with respect to a polypeptide (e.g., a chemokine or antigen) as used herein refers to a polypeptide sequence comprising the full length amino acid sequence of the polypeptide, or comprising a fragment, analogue, derivative or truncation isoform of the polypeptide. Functional equivalents also include, for example, the polypeptide, or its fragment, analogue, derivative or truncation isoform, in salt, complex, or analog form. Functional equivalents retain some or all of the biological activity of the corresponding polypeptide. Where the polypeptide referred to is an antigen, xe2x80x9cbiological activityxe2x80x9d refers to the ability of the functional equivalent to bind to an antibody that will also bind to the native antigen.
The terms xe2x80x9cpeptide,xe2x80x9d xe2x80x9cpolypeptide,xe2x80x9d and xe2x80x9cproteinxe2x80x9d are used interchangeably herein and are intended to refer to amino acid sequences of any length.